Identifying the Molecular Pathways Regulating Glucose-dependent Insulin Secretion

确定调节葡萄糖依赖性胰岛素分泌的分子途径

• 批准号: 8408842
• 负责人: David Altshuler
• 金额: $ 4.35万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8408842
• 关键词: 2-cyclopentyl-5-(5-isoquinolylsulfonyl)-6-nitro-1H-benzo(D)imidazole; Affect; Agonist; American; Beta Cell; Biological; Biological Assay; Biology; Blood; Cell Line; Cell physiology; Cells; Characteristics; Chemicals; Chronic; Collaborations; Defect; Dependence; Development; Diabetes Mellitus; Diagnosis; Disease; Dose; Ensure; Environment; Enzyme-Linked Immunosorbent Assay; Epidemic; Exhibits; Extravasation; FDA approved; Functional disorder; Generations; Glucose; Goals; Human; Human Genetics; Hypoglycemia; Inflammation; Inherited; Inhibitory Concentration 50; Institutes; Insulin; Lead; Luciferases; Measures; Membrane Potentials; Molecular; Molecular Probes; Mus; Nature; Non-Insulin-Dependent Diabetes Mellitus; Observational Study; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Physiological; Play; Prediabetes syndrome; Predisposing Factor; Protocols documentation; Proxy; Research; Research Personnel; Resistance; Risk; Role; Secretory Cell; Secretory Vesicles; Stress; Structure of beta Cell of islet; Structure-Activity Relationship; Testing; Therapeutic; Therapeutic Agents; Variant; Work; base; burnout; compliance behavior; enzyme activity; follow-up; global health; glucose metabolism; high throughput screening; impaired glucose tolerance; improved; incretin hormone; inhibitor/antagonist; insulin secretion; islet; mitochondrial membrane; novel; prevent; programs; response; small molecule; sugar; therapeutic development; tool

DESCRIPTION (provided by applicant): Beta-cell dysfunction plays a central role in the pathogenesis of type 2 diabetes (T2D).1 Even prior to diagnosis, patients with impaired glucose tolerance (i.e., "prediabetes") lack the first phase of glucose- stimulated insulin secretion and exhibit resistance to the amplifying effect of incretin hormones that normally prime the beta cell at meals.2 While deficient insulin secretion is now recognized as a prerequisite for both the onset and progression of T2D, the molecular pathways regulating this crucial physiologic response remain largely unknown. Moreover, no treatments are currently available to prevent the progressive nature of beta-cell decline characteristic of diabetes,3 and no small molecules exists that are FDA-approved to directly increase insulin secretion in a glucose-dependent manner, thereby avoiding the risk of hypoglycemia.4 To develop chemical probes of the molecular pathways regulating glucose-dependent insulin secretion and to identify small-molecule leads for glucose-dependent therapeutics, our proposed project wil employ a novel insulin secretion bioassay in the setting of near-stimulatory glucose conditions. Historically, a screen to identify modulators of insulin secretion has not been feasible, due to the lack of a suitable functional readout; the standard insulin ELISA is labor intensive, expensive, and limited to 96-well format. To overcome this bottleneck, we developed a high-throughput luminescent insulin secretion assay in which luciferase is targeted to the secretory vesicles of a beta cell an co-secreted with insulin upon stimulation. Luciferase serves as a close proxy for insulin, with enzyme activity responding appropriately to known secretagogues and inhibitors of insulin secretion, and in close correlation (r2 = 0.96) with insulin as measured by ELISA. Compounds found to increase luciferase secretion in near-stimulatory glucose concentrations will be counter- screened in the absence of glucose, to identify those exhibiting glucose-dependent effects. The activity of such compounds will be confirmed in a secondary assay using an insulin ELISA. Thereafter, the affected pathways within the beta cell will be explored using assays for ATP level and mitochondrial membrane potential. Lastly, top hits will be tested for their effect on dissociated human islets to confirm cross-species relevance. If successful, this phenotypic screen will identify new probes of pathways modulating beta-cell function in a glucose-dependent manner, improving our understanding of a causal disease mechanism for type 2 diabetes. In addition, this valuable toolbox of small molecules should prove useful to researchers exploring the response of the beta cell to known pathogenic insults, including ER stress, inflammation and glucolipotoxicity. Lastly, our screen may identify lead compounds for the development of therapeutics to safely treat diabetes without risk of hypoglycemia.

描述（由申请人提供）：β细胞功能障碍在2型糖尿病的发病机理中起着核心作用（T2D）。1甚至在诊断前，葡萄糖耐受性受损的患者（即“糖尿病率”）缺乏葡萄糖刺激的胰岛素分泌和表现出良性效应的葡萄糖的第一阶段，缺乏葡萄糖的第一阶段现在，缺乏胰岛素的分泌被认为是T2D发作和进展的先决条件，调节这种关键生理反应的分子途径仍然在很大程度上未知。此外，目前尚无治疗可预防糖尿病的β细胞下降特征的渐进性，3和不存在的小分子存在于FDA批准的情况下直接增加胰岛素的分泌，以葡萄糖依赖性依赖性依赖性葡萄糖，从而避免了低糖的风险。葡萄糖依赖性疗法，我们提出的项目将在近刺激性葡萄糖条件下采用新型的胰岛素分泌生物测定。从历史上看，由于缺乏合适的功能读数，鉴定胰岛素分泌调节剂的屏幕是不可行的。标准的胰岛素ELISA是劳动密集型，昂贵且限制为96孔格式的。为了克服这种瓶颈，我们开发了一种高通量发光胰岛素分泌测定法，其中荧光素酶靶向β细胞的分泌囊泡，刺激后与胰岛素共凝聚。荧光素酶是胰岛素的紧密替代，其酶活性适当地响应已知的促促分泌物和胰岛素分泌的抑制剂，并与ELISA测量的胰岛素紧密相关（R2 = 0.96）。在没有葡萄糖的情况下，将对近刺激性葡萄糖浓度中的荧光素酶分泌的化合物进行筛选，以鉴定出那些表现出葡萄糖依赖性作用的人。这种化合物的活性将在次级测定中使用胰岛素ELISA确认。此后，将使用ATP水平和线粒体膜电位的测定探索Beta细胞内的受影响途径。最后，将测试最高命中的作用 解离的人类胰岛确认跨物种相关性。如果成功，此表型筛选将以葡萄糖依赖性方式识别调节β细胞功能的新探针，从而提高我们对2型糖尿病因果疾病机制的理解。此外，这种有价值的小分子工具箱应证明对探索β细胞对已知致病性损伤的反应的研究人员有用，包括ER应激，炎症和糖脂肪毒性。最后，我们的屏幕可能会鉴定出开发治疗剂的铅化合物，以安全地治疗糖尿病而没有血糖风险。